Immortal cell line derived from grouper Epinephelus coioides and its applications therein

ABSTRACT

The present invention describes (1) an immortal cell line derived from grouper and a method for establishing the cell line; (2) methods for mass producing and purifying aquatic 10 viruses using the immortal cell line from grouper; (3) an anti-NNV antibody and a method for producing the anti-NNV antibody; and (4) a vaccine of NNV and a method for protecting fish against NNV infection. The present immortal cell line is derived from the grouper and is susceptible to the viral families of Birnaviridae such as Infectious Pancreatic Necrosis Virus (IPNV); Herpesviridae such as Eel Herpes Virus Formosa (EHVF); Reoviridae such as Hard Clam Reovirus (HCRV); and Nodaviridae such as Nervous Necrosis Virus (NNV).

RELATED APPLICATION

[0001] This application claims the priority of U.S. ProvisionalApplication No. 60/110,699, filed on Dec. 3, 1998, which is incorporatedherein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to an immortal cell line (GF-1)derived from the fin tissue of grouper Epinephelus coioides and themethod of establishing the GF-1 cell line. The GF-1 cell line issusceptible to a number of aquatic viruses, including, but not limitedto, Infectious Pancreatic Necrosis Virus (IPNV), Eel Herpes VirusFormosa (EHVF), and Nervous Necrosis Virus (NNV). This invention alsorelates to the method of mass producing and purifying the aquaticviruses using an immortal cell line from grouper such as the GF-1 cellline as a host. Additionally, this invention relates to an anti-NNVantibody and the method of producing the anti-NNV antibody. Finally,this invention relates to a vaccine of NNV and the method for protectingfish against NNV infection.

BACKGROUND OF THE INVENTION

[0003] Nervous necrosis virus (NNV), a pathogen found in many varietiesof hatchery-reared marine fish, has caused mass mortality of such fishat their larval or juvenile stages. NNV belongs to the familyNodaviridae. Fish nodaviruses isolated from different species (such asSJNNV, BFNNV, JFNNV, TPNNV, RGNNV, GNNV etc.) are closely related toeach other owing to the high similarity of the conserved region of theircoat protein genes. NNV, also named as fish encephalitis virus (FEV) andpiscine neuropathy nodavirus (PNN), is an unenveloped spherical viruswith particles sized between 25 and 34 nm. The virus is characterized byvacuolation of the nerve tissues. Viral Nervous Necrosis (VNN) diseasehas been found in many countries under various names such as viral fishencephalitis, fish encephalomyelitis, cardiac myopathy syndrome. Thehosts of NNV include many species of marine fish, for example;parrotfish, sea bass, turbot, grouper, stripped jack, tiger puffer,berfin flounder, halibut, barramundi, and spotted wolffish.

[0004] According to the statistics shown in 1993, approximately 159 fishcell lines have been established which have demonstrated a capacity forgrowing fish viruses (Fryer and Lannan, J. Tissue Culture Method (1994),10:57-94). Most of these cell lines are derived from the tissues offreshwater fish. There are only thirty-four cell lines which areoriginated from marine fish. Although some of the fish cell lines, whichinclude RTG-2, CHSE-214, BF2, SBL, FHM, EPC, have been tested for thesusceptibility of fish nodavirus, none of these cells lines has showncytopathic effects (CPE) after viral inoculations.

[0005] In 1996, SSN-1 cell line, a cell line derived from stripedsnakehead Channa Striatus, has been successfully used for isolating seabass nodavirus (Frerichs et al., J. General Virology (1996)77:20672071).However, SSN-1 cell line has been known to be persistently contaminatedwith C-type retrovirus (Frerichs et al., J. General Virology (1991)72:2537-2539). Therefore, it is not suitable for the production of fishnodavirus.

[0006] Viral diseases cannot be cured by therapeutic reagents. The bestways to contain viral diseases include prevention through earlydetection and the development of vaccines. In either way, theunderstanding of the biological, biochemical, and serologicalcharacteristics of the virus is fundamentally required, which in turnrelies on the industry to have the capacity of mass producing the pureform of viruses, preferably through an in vitro cell culture system.Therefore, the development of a new cell line which can be susceptibleto fish nodavirus is desperately in demand in order to control the widespread of fish viral diseases due to fish nodavirus infection.

[0007] Grouper is an important hatchery fish in Taiwan. In recent years,there have been several reports regarding the establishment of celllines derived from grouper. For example, Chen et. al. (Japan ScientificSociety Press (Tokyo) (1988) 218-227) have reported their establishmentof several cell lines from the fin and kidney tissues of grouperEpinephelus awoara. Lee (Master Thesis from the Department of Zoology atthe National Taiwan University, 1993) also has reported hisestablishment of the cell lines derived from the eye pigment cells andbrain tissue of grouper Epinephelus amblycephalus. However, Chen et al.do not provide sufficient data in support of the claim for immortalityin their cell lines and Lee expressly indicates in his thesis that hisgrouper cell lines are not immortal. Moreover, neither Chen et al.'s norLee's cell lines are susceptible to fish nodavirus.

[0008] Recently, severe mortality among groupers has repeatedly occurredwhich is caused primarily by nodavirus. As present, fish nodavirus hasbeen discovered in grouper and can be isolated from moribund grouperwhich possess symptoms of VNN disease (Chi et al., J. Fish Disease(1997) 20:185-193). Electron microscopic examination of the tissues fromgrouper shows that, in addition to nodavirus infection, grouper issusceptible to other viral infections (Chi, COA Fisheries Series No.61,Reports on Fish Disease Research (1997) 18:59-69). The fact that someviruses have host specificity makes a cell line derived from groupermore appropriate for investigating the specific viruses isolated fromgrouper.

[0009] In the invention to be presented below, an immortal cell linederived from the fin tissue of grouper Epinephelus coioides (Hamilton)will be introduced: The cell line of the present invention issusceptible to various viruses, particularly fish nodavirus such asGNNV. Using the present cell line, various aquatic viruses can be massproduced and purified. The purified viruses are useful for antibody andvaccine production to protect fish from viral infections.

SUMMARY OF THE INVENTION

[0010] A first embodiment of the present invention provides for animmortal cell line derived from grouper, preferably, an immortal cellline (GF-1) which is derived from the fin tissue of grouper Epinepheluscoloides. GF-1 is susceptible to, and can mass produce viruses whichinclude, but are not limited to, viruses from the families ofBirnaviridae (such as infectious pancreatic necrosis virus [IPNV]),Herpesviridae (such as eel herpes virus Formosa [EHVF]), Reoviridae(such as hard clam reovirus [HCRV]), and Nodaviridae (such as groupernervous necrosis virus [GNNV]).

[0011] The first embodiment also provides for a method of establishingan immortal cell line. The method comprises the steps of: (1)establishing a primary cell culture by placing cells released from thefin tissue of grouper Epinephelus coioides in a tissue culture flask toform a monolayer of cells; (2) subculturing and maintaining themonolayer of cells in a media suitable for cell subculturing; and (3)monitoring a transformation of cells which is characterized by a changein chromosome number distribution, plating efficiency, fetal bovineserum (FBS) requirement, and susceptibility to aquatic viruses,particularly fish nodavirus such as GNNV.

[0012] A second embodiment of the invention provides for a method forgrowing a virus using the immortal cell line derived from grouper,preferably the GF-1 cell line. The method comprises the steps of. (1)inoculating the virus into the cell line; and (2) incubating the cellline in a nutrient medium suitable for growth and replication of thevirus. The viruses which are susceptible to and can be replicated in theimmortal cell line include viruses from the families of Birnaviridae,Herpesviridae, Reoviridae, and Nodaviridae, and, in particular, lPNV ofBirnaviridae, EHVF of Herpesviridae, HCRV of Reoviridae, and GNNV ofNodaviridae.

[0013] The second embodiment also provides for methods of mass producingthe viruses using the immortal grouper cell line, purifying the viruses,and detecting the viruses in the cell line. The method for massproducing the virus comprises: (1) inoculating the virus into thegrouper cell line; (2) incubating the cell line in a nutrient mediumsuitable for growth and replication of the virus; and (3) harvesting thevirus from the cell line.

[0014] The method for purifying a virus comprises: (1) inoculating thevirus into the grouper cell line; (2) incubating the cell line in anutrient medium suitable for growth and replication of the virus untilthe appearance of cytopathic effects (CPE); (3) harvesting the virusfrom the cell line; and (4) purifying the virus using density gradientcentrifugation. The preferable density gradient is a CsCl densitygradient. However, other density gradients which yield a sufficientvirus harvest are also within the scope of the invention.

[0015] The present method for detecting a virus in the immortal groupercell line comprises: observing a development of cytopathic effects (CPE)in the cell line under microscope. The virus can be further confirmed bythe electron microscopic method which comprises the steps of. (1) fixingthe cell line in glutaraldehyde and osmium tetraoxide; (2) performingultrathin sectioning of the fixed cells; and (3) detecting viralparticles in the ultrathin section of the fixed cell line under anelectron microscope.

[0016] There are four methods which contribute to the specific detectionof NNV in the immortal grouper cell line after the CPE is detected inthe cell line. A first method uses the polymerase chain reaction (PCR),which comprises the steps of: (1) extracting a viral RNA from the cellline; (2) amplifying the viral RNA by PCR using a reverse primer (SEQ IDNO. 1) and a forward primer (SEQ ID NO. 2). A second method uses awestern immunoblot, which comprises the steps of. (1) extracting theviral protein from the cell line; (2) electrophoresizing the viralprotein in an SDS-polyacrylamide gel; and analyzing the polyacrylamidegel by western immunoblot using an anti-NNV serum. A third method usesan enzyme-linked immunosorbent assay (ELISA) method to detect NNVprotein. A fourth method uses immunofluorescent staining by couplingfluorescein isothiocyanate (FITC) conjugated goat anti-mouse antibodieswith mouse anti-NNV serum to thereby detect NNV within the cells.

[0017] A third embodiment of the invention provides for an anti-NNVantibody and a method of making the anti-NNV antibody. The anti-NNVantibody is preferably a monoclonal antibody. The anti-NNV antibody isprepared by administering an effective amount of NNV to a suitableanimal, preferably a mouse or a rabbit, to stimulate an immunoresponsein the animal. The NNV used for making the anti-NNV antibody ispreferably the one harvested from the grouper cell line and furtherpurified by a CsCl density gradient centrifugation (NNV has a buoyantdensity of approximately 1.34 g/ml in CsCl).

[0018] A fourth embodiment of the invention provides for a vaccine toNNV and a method for protecting fish against NNV infection. The NNVvaccine comprises an immunogenically effective amount of killed NNV. Thevaccine further comprises a material selected from the group consistingof adjuvants, plasticizers, pharmaceutical excipients, diluents,carriers, binders, lubricants, glidants and aesthetic compounds, andcombinations thereof. The vaccine can be administered orally or byinjection. Oral administration is the preferred method of vaccinatingfish. For the orally administered vaccine, an enteric coating which isimpervious to dissolution in the stomach of fish can be added to thevaccine. The NNV useful for making the anti-NNV antibody is preferablythe one harvested from the grouper cell line and further purified by aCsCl density gradient centrifugation (NNV has a buoyant density ofapproximately 1.34 g/ml in CsCl). The NNV is preferably inactivated. Thepresent method for protecting fish against NNV infection comprises:administrating an effective amount of the NNV vaccine to a fish.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 shows the morphology of the GF-I cells observed under aninverted microscope. (A) A semi-confluent monolayer where bothfibroblast-like and epithelial cells co-existed, and (B) a confluentmonolayer of GF-1 cells at subculture 80 where fibroblast-like cellswere the predominant cells. In the figure, an arrowhead indicatesfibroblast-like cells. Similarly, an arrow indicates epitheloid cells.Bar=10 μm

[0020]FIG. 2 shows the chromosome number distribution of the GF-1 cellsat (A) subculture 50, and (B) subculture 80.

[0021]FIG. 3 shows the effect of fetal bovine serum (FBS) on the growthrate of GF-1 cells at (A) subculture 50, and (B) subculture 80.

[0022]FIG. 4 shows the effect of temperature on the growth rate of GF-1cells at subculture 80.

[0023]FIG. 5 shows the cytopathic effects (CPE) of GF-1 cells atsubculture 80 after infection by (A) IPNV AB strain, (B) IPNV SP strain,(C) IPNV VR299 strain, (D) IPNV EVE strain, (E) HCRV, (F) fishnodavirus_GNNV isolate, and (G) HEVF, as compared with (H) UninfectedGF-1 cells.

[0024]FIG. 6 shows the agarose gel electrophoresis of the product byRT-PCR amplification using a pair of primers (SEQ ID NO: 1 and SEQ IDNO: 2) specific to the target region T4 of fish nodavirus SJNNV. Lane 1,PCR product from GNNV-infected GF-1 cells; lane 2, PCR product fromnon-infected GF-1 cells. M: pGEM marker.

[0025]FIG. 7 is an electron micrograph of GNNV-infected GF-1 cells.Inclusion bodies (indicated by arrowhead) and numerous non-envelopedviral particles are shown in the cytoplasm. I: an inclusion body filledwith viral particles. M: mitochondria, N: nucleus. Arrowhead indicatesthe viral particle. Bar=1 μm.

DETAILED DESCRIPTION OF THE INVENTION

[0026] In accordance with a first embodiment of the present invention,there is provided an immortal cell line which is derived from grouper,preferably from the grouper Epinephelus coioides, and more preferablyfrom the fin tissue of grouper Epinephelus coioides. A vital sample ofthe immortal cell line derived from the fin tissue of grouperEpinephelus coioides, the GF-1 cell line, is intended to be deposited atthe American Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110-2209, before the issuance of the patent, under theprovisions of the Budapest Treaty for the International Recognition ofthe Deposit of Microorganisms for the Purpose of Patent Procedure. Theassigned deposit number for this cell line is ATCC No.______

[0027] This embodiment also provides for a method of establishing animmortal cell line. The experimental designs and results pertaining tothe establishment of an immortal cell line are illustrated, but notlimited to, in the following examples:

EXAMPLE 1 Establishment of the GF-1 Cell Line

[0028] The GF-1 cell line was established and maintained as follows:

[0029] (1) Primary Culture

[0030] A grouper (Epinephelus coioides, Hamilton) weighing 1 kg was usedfor the establishment of the primary culture. The fish was dipped in 5%chlorex for 5 min, and then wiped with 70% alcohol. The fin wasdissected from the body, and washed three times in a washing medium(containing L15 plus 400 IU/ml of penicillin, 400 μg/ml of streptomycinand 10 μg/ml of fungizone). After washing, the fin tissue was mincedwith scissors and then placed into 0.25% trypsin solution (0.25% trypsinand 0.2% EDTA in phosphate-buffered saline [PBS]). The tissue fragmentsin trypsin solution were slowly agitated with a magnetic stirrer at 4°C. At 30 min intervals, cells released from the tissue fragment werecollected by centrifugation. Next, cells were re-suspended in a completemedium (containing L15 plus 20% of fetal bovine serum [FB-S], 100 IU/mlof penicillin, 100 μg/ml of streptomycin, and 2.5 μg/ml of fungizone),transferred into a 25 cm² tissue culture flask and, finally, cultured at28° C.

[0031] (2) Subculture and Maintenance

[0032] When the confluent monolayer of cells had formed in the primaryculture, cells were dislodged from the flask surface by treating with0.1% trypsin solution (containing 0.1% of tryspin and 0.2% of EDTA inPBS). The released cells were then transferred into two new flaskscontaining fresh L15 medium plus 20% of FBS. Cells were subcultured at asplit ratio of 1:2. For the first ten subcultures of the GF-1 cells, aconditioned medium consisting of 50% old and 50% fresh medium was used.The concentration of FBS in the maintaining L15 medium was 10% forsubcultures 11-70, and decreased to 5% after subcultures 70. Also,during the first twenty passages, GF-1 cells were subcultured at a 9-dayinterval. For the next 21^(th)-70^(th) passages, the GF-1 cells weresubcultured at a 5-day interval. After 71 passages, GF-1 cells weresubcultured at a 3 -day interval.

[0033] (3) Test for Mycoplasma Contamination in the GF-1 Cell line

[0034] The GF-1 cell line was propagated for three transfers inantibiotic-free L15-10% FBS and tested for the presence of bacteria,fungi, and mycoplasma. A mycoplasma stain kit (Flow Laboratories,U.S.A.) was used for mycoplasma testing.

[0035] (4) Test for the Viability of the GF-1 Cell Line

[0036] The viability of the GF-1 cell line was tested by first removingthe cells from the flask. Then, the cells were separated from the mediumby centrifugation, and re-suspended in a freezing medium consisting of10% dimethyl sulfoxide (DMSO) and 90% FBS. Ampules (NUNC, Denmark)containing 5×10⁶ cells/ml /ampule were held at −20° C. for one hour,followed by staying at −70° C. overnight before being transferred toliquid nitrogen (−176° C.). After one month and one year, the ampuleswere thawed in a 30° C. water bath. The cells were separated from thefreezing medium by centrifugation. The cells were re-suspended inL15-10% FBS. The viable cells were determined by trypan blue staining.The number of cells was counted using a hemacytometer. The thawed cellswere re-seeded into a 25 cm² flask for further observation.

[0037] (5) Chromosome Number Distribution

[0038] The distribution of the chromosome numbers in GF-1 cells atsubculture 50 and subculture 80 were studied using semi-confluent andactively growing cells. Cells were pre-treated with 0.1 μg/ml Colcemid(Gibco, Grand Island, N.Y.) for 5 hours at 28° C. before being dislodgedwith 0.1% of trypsin solution. After centrifugation at 1000 g for 10min, the cells were re-suspended in a hypotonic solution (containing 8parts of distilled water and 1 part of PBS) for 30 min. The cells werethen partially fixed by adding several drops of Carnoy fixative(containing 1 part of Glacial acetic acid and 3 parts of 100% methanol).The partially fixed cells were further centrifuged at 800 g for 10 minat 4° C. The supernatant was discarded, and the cells were fixed infresh, cold Carnoy fixative for 20 min. The suspension of fixed cellswas dropped onto a 76×26 mm slide. The slide was air-dried and the cellswere stained with 0.4% Giemsa stain (Sigma, St. Louis, Mo., USA) for 30min. The chromosome numbers were observed and counted under an OlympusVanox microscope.

[0039] (6) Plating Efficiency

[0040] The plating efficiency of the GF-1 cells was estimated atsubcultures 50 and 80. Cells were seeded into a 25 cm² flask at adensity of 100 cells per flask. Following 15 days of incubation, themedium was removed and the cell colonies were fixed with 70% ethanol andstained with 0.4% Giemsa. The colonies in each flask were then countedusing an Olympus IM inverted microscope. Carp fin (CF), black porgyspleen (BPS-1), tilapia ovary (TO-2) and eel kidney (EK) cell lines wereplated the same way as the GF-1 cell line for comparison purpose.

[0041] (7) Effects of FBS Concentration and Temperature on the Growth ofthe GF-1 Cells

[0042] The effects of the concentration of FBS on GF-1 cell growth weredetermined at subcultures 50 and 80. Two replicates were prepared foreach FBS concentration. At selected intervals, two flasks were withdrawnfrom each concentration of FBS, and the mean number of cells wascounted.

[0043] To determine the effects of temperature on the growth of GF-1cells at subculture 80, replicated cell cultures in 25 cm² flaskscontaining L15-10% FBS were incubated at 18° C., 28° C. and 35° C. Themean number of the GF-1 cells from two replicated flasks at eachtemperature was counted at selected intervals.

[0044] Results

[0045] Primary Culture and Subculture of the GF-1 Cells

[0046] A monolayer of cells was formed in the primary cultureapproximately two weeks after the implantation. Fibroblast-like cellsand epitheloid cells co-exist in the cell population (FIG. 1). The GF-1cells have been successfully subcultured for more than 160 times since1995, subsequently becoming a continuous cell line.

[0047] The GF-1 cells were subcultured at 9-day intervals in L15-20% ofFBS during the first twenty subcultures, at 5-day intervals in L15-10%of FBS during the 21^(st)-70^(th) subcultures, and at 3-day intervals inL15-5% of FBS since subculture 71. Contact inhibition of the GF-1 cellswas found in cultures before subculture 50, and gradually decreasedbetween subculture 51 and 80.

[0048] The viability of the GF-1 cells at subculture 80 after one yearand one month was 73%. The re-seeded cells grew readily when incubatedat 28° C. in L15-5% of FBS.

[0049] Chromosome Number

[0050] The chromosome number of the GF-1 cells at subculture 50 wasdistributed between 7 and 44 with the mode set at 32 (FIG. 2A). Thechromosome number of the GF-1 cells at subculture 80 was distributedbetween 17 and 42 in 100 cells examined, and had a bimodal distributionwith modes set at 32 and 36 (FIG. 2B). Both micro- and macro-chromosomeswere found in metaphase-arrested cells.

[0051] Plating Efficiency

[0052] The plating efficiency of the GF-1 cells seeded at a density of100 cells/ flask was 21% at subculture 50 which increased to 80% atsubculture 80. In comparison, the plating efficiencies of CF, BPS-1,TO-2 and EK cell lines seeded at a density of 100 cells/flask were 22%,13%, 48%, and 63%, respectively. The increase in plating efficiency inGF-1 cells suggests the occurrence of transformation during subcultures50-80.

[0053] Effects of FBS Concentration and Temperature on the Growth of theGF-1 Cells

[0054]FIG. 3 illustrates the effects of FBS concentration on the growthof the GF-1 cells at subcultures 50 and 80. The growth of the GF-1 cellsat both subcultures 50 and 80 corresponded to the concentration of FBS,i.e., the higher the FBS concentration, the greater the growth of cells.However, when the growth rates of the GF-1 cells at subcultures 50 and80 were compared, the GF-1 cells at subculture 80 demonstrated a muchgreater growth potential than those at subculture 50, especially whenthe FBS concentrations were at 2%, 5%, and 10%. For example, at day 4 ofthe cell cultures containing 10% of FBS, the GF-1 cells at subculture 50have 3.5×10⁶ cells/25 cm² flask, whereas the GF-1 cells at subculture 80have 5.0×10⁶ cells/25 cm² flask. These results suggest that therequirement of FBS for cell growth decreased at subculture 80, which isan indication that the transformation of cells had occurred during theperiod from subculture 50 to subculture 80.

[0055]FIG. 4 illustrates the effect of temperature on the growth of theGF-1 cells at subculture 80. The results show that the GF-1 cells grewwell at 28° C. and 35° C. However, the growth of the GF-1 cells culturedat 35° C. started to decline at day 4, suggesting that maintaining thecell culture at 35° C. may have long-term effects on cell growth. TheGF-1 cells did not grow well at 18° C.

[0056] In accordance with a second embodiment of the present invention,there are provided methods for producing the aquatic viruses in the GF-1cells, purifying the viruses, and detecting a virus in the immortal cellline. The experimental designs pertaining to this embodiment areillustrated as follows:

EXAMPLE 2 Methods For Producing Viruses Using the GF-1 Cell Line andMethods for Detecting the Viruses in the Cell Line

[0057] (1) Test for Susceptibility of the GF-1 Cells to Aquatic Viruses

[0058] Infectious pancreatic necrosis virus (IPNV, strain AB, SP, VR299and EVE), hard clam reovirus (HCRV), eel herpes virus Formosa (EHVF) andnervous necrosis virus (NNV, GNNV isolate) were used to infect the GF-1cells at subculture 80. The susceptibility to GNNV was also examined inBGF-1 cell line, which was derived from the fin of the banded grouperEpinephelus awoara.

[0059] Each of the monolayer GF-1 cells was inoculated with 0.5 ml ofvarious aquatic virus with titer of 10³ TCID₅₀ /0.1 ml. After a 30-minadsorption period, the cells from each flask were washed three timeswith PBS, followed by the addition of 5 ml of L15-2% FBS to each flask.The flasks were then incubated separately at 20° C. and 28° C. Thesupernatants of culture cells were collected and titrated for 6 dayspost viral infection.

[0060] (2) Multiplication and Purification of Aquatic Viruses in theGE-1 Cell Line

[0061] Viral isolate was inoculated at an MOI (multiplicity ofinfection) of 0.01 into the GF-1 cell line. When CPE appeared, the GF-1cells were scraped into the medium and the cell debris was pelleted at10000×g for 30 min (the first pellet). The supernatant was transferredto a bottle and polyethylene glycol (PEG, molecular weight 20000) andNaCl were added to reach a final concentration of 5% and 2.2%separately. The supernatant was then stirred for 4-6 hours at 4° C., andthe virus particles were pelleted by centrifugation at 10000×g for 1hour (the second pellet). The first pellet and the second pellet werere-suspended in a small amount of TNE buffer (0.1M Tris, 0.1M NaCl, 1 mMEDTA, pH 7.3), to which an equal volume of Freon 113 was added. Themixture was shaken vigorously for 5 min, and the emulsion was separatedinto the Freon and aqueous phase by centrifugation at 3000×g 10 min. Theaqueous phase was collected, layered on a preformed 10-40% (w/w) CsClgradient, and centrifuged at 160000×g for 20 hours. The visible virusband was collected, diluted with 10 ml of TNE buffer, and pelleted againby centrifugation at 150,000 g for 1 hours. The final pellet wasresuspended in a small volume of TE buffer (0.1 M Tris, 1 mM EDTA, pH7.3).

[0062] (3) Detection of Aquatic Viruses in the GF-1 Cell Line

[0063] In general, when a virus infects a cell line which is susceptibleto the virus, a CPE of the cell culture can be observed within a coupleof days after the infection. The appearance of CPE serves as evidencethat the virus has successfully infected and multiplied in the cellline. The viral infection in the cell line can be further confirmedusing an electron microscopic technique which is described as follows:The virus-infected cells were fixed in 2.5% glutaraldehyde in 0.1M ofphosphate buffer at pH 7.4 and post-fixed in 1% of osmium tetraoxide.The cells were ultrathin sectioned. The ultrathin sections were stainedwith uranyl acetate-lead citrate and examined under a Hitachi H-600Aelectron microscope. The viral particles should appear as homogeneous,spherical particles in the cytoplasm of the cells.

[0064] There are also three methods which are directed to specificdetection of NNV in the GF-1 cell line:

[0065] (A)Detection of NNV in the GF-1 Cells by Polymerase ChainReaction (PCR) Amplification

[0066] A PCR amplification method was used to confirm that the GF-1cells are able to proliferate NNV. The method required that the viralRNA be extracted from the supernatant of the NNV-infected cells afterCPE appeared using a Rneasy™ mini kit (QIAGEN). For reversetranscription, extracted viral RNA was incubated at 42° C. for 30 min in40 □l of 2.5×PCR buffer (25 mM of Tris-HCl, pH 8.8, 3.75 mM of MgCl₂,125 mM of KCl, and 0.25% of Triton X-100) containing 2 U of MMLV reversetranscriptase (Promega), 0.4 U of RNsin (Promega), 0.25 mM of dNTP, and0.5 □M of the reverse primer R3 (5′ CGAGTCAACACGGGTGAAGA 3′) (SEQ ID NO.1). Following the cDNA synthesis, 40 □l of the cDNA mixture were diluted2.5-fold with diethyl pyrocarbonate (DEPC)-treated H₂O (containing 0.025U of DNA polymerase [Biometra], 0.1 mM of dNTP and 0.5 □M of the forwardprimer F2 [5′ CGTGTCAGTCATGTGTCGCT 3′] [SEQ ID NO. 2]), and incubated inan automatic thermal cycler (TouchDown™ thermal cycler, Hybaid company).The target region for the primer set (F2, R3) is T4 (400 bp). The PCRproducts corresponding to T2 and T4 were amplified from the nucleicacids of NNV-infected GF-1 cells.

[0067] (B) Detection of NNV in the GF-1 Cells by Western Immunoblot

[0068] A western immunoblot method was used to specifically detect theNNV proteins. The viral sample was prepared as follows: NNV wasinoculated into the GF-1 cells and incubated at 20-32° C. After 5 daysof incubation, the NNV-infected cells were pelleted by centrifugation at1000 g for 10 min. The cell pellets were loaded onto a 10%SDS-polyacrylamide gel. After electrophoresis, the proteins were blottedto an immobilon-P transfer membrane (Millipore), which was then soakedin a 3% skim milk tris buffered saline (TBS) for 1 hr. The membrane wasthen incubated with an antiserum against NNV for 1 hr at roomtemperature, washed with TBS, reacted with a peroxidase-conjugate goatsystem for 1 hr, and stained with a substrate containing 6 mg of4-chloronaphthol in 20 ml of methanol and 60 □l of H₂O₂ in 100 ml ofTBS.

[0069] (C) Detection of NNV in the GF-1 Cells by Enzyme-LinkedImmunoabsorbent Assay (ELISA)

[0070] ELISA is an immunological method which uses an enzyme-labeledimmunoreactant (antigen or antibody) and an immunosorbent (antigen orantibody bound to a solid) to identify specific serum or tissueantibodies or antigens. The ELISA test was conducted as follows: aneffective amount of purified NNV proteins was coated onto a microtiterplate at 4° C. overnight. Then, 3% of bovine serum albumin (BSA) wasadded to the plate (used as blocking agent) and incubated at 37° C. for1 hr. The plate was then washed 3 times with buffer. Next, a dilutedrabbit anti-NNV serum was added to the plate and incubated at 37° C. for1 hr. This was followed by the addition of goat anti-rabbitIgG-horseradish peroxidase serum at 37° C. for 1 hr and3,3′,5,5′-tetramethyl benzidine was added for color development. Thecolor reaction was stopped with 1 N H₂SO₄. The optical density of thewells in the microtiter plate was measured at 450 nm with an ELISAreader (Dynatech MR 5000).

[0071] (D) Detection of NNV in the GF-1 Cells by ImmunofluorescentStaining

[0072] To detect the virus that proliferated in the GF-1 cells, cellcultures were fixed by 10% formalin for 12 hrs after viral infection.The fixed cell cultures were treated with 0.2% of Triton X-100 andwashed with PBST (phosphate buffer with 0.05% Tween 20). TheTriton-treated cell cultures were further washed with 3% of skim milk asblocking agent and then reacted with mouse anti-NNV serum. Finally, theantibody-treated cell cultures were stained with fluoresceinisothiocyanate (FITC) conjugated goat anti-mouse antibodies.

[0073] Results

[0074] Table 1 summarizes the results of virus susceptibilities of theGF-1 cells to IPNV (AB, SP, VR299, EVE strains), HCRV, EHVF and NNV(GNNV isolate), which were determined by first observing the appearanceof CPE in the cells after the viral inoculation, followed by thedetermination of viral titers (TCID₅₀/ml). TABLE 1 ViralSusceptibilities of GF-1 Cells at Subculture 80 Initial Viral VirusYield/ml Cell Inoculum CPE (TCID₅₀/ml) line Virus (TCID₅₀) 28° C. 20° C.28° C. 20° C. GF IPNV AB 10³ − + ND 10^(9.5) SP 10³ − + ND  10^(10.8)VR299 10³ − + ND 10^(9.8) EVE 10³ − + ND 10^(9.6) HCRV 10³ − + ND 10^(11.0) EHVF 10³ + + 10^(8.1) 10^(7.0) GNNV 10³ + − 10^(8.3) ND

[0075] As shown in Table 1, for the IPNV strains and HCRV, CPE appearonly when the cells are incubated at 20° C. For EHVF, CPE appears atboth 20° C. and 28° C. However, for GNNV, CPE appears at 28° C. Theyields of the viruses in GF-1 cells at subculture 80, which are rangedbetween 10^(7.0) (as for EHVF at 20° C.) and 10^(11.0) (as for HCRV at20° C.), are extremely high.

[0076] Typically, for an aquatic virus such as GNNV, CPE began at the3rd day of infection when some rounded, granular, refractile cells beganto appear in the cell culture (FIG. 5). Soon more and more cells becameround and swollen. The swollen cells became larger and finally startedto detach from the cell culture and float in the culture media. Most ofthe detached cells were completely disintegrated. The culture fluid fromcell culture showing CPE could transmit other GF-1 cells. Thisexperiment also tested the susceptibility of BGF-1 cell line (derivedfrom the fin of the banded grouper Epinephelus awoara) to GNNV. Theresults showed that no CPE was found after the viral infection.

[0077] Typically, for an aquatic virus such as GNNV, the virus could beobserved in the cytoplasm of the GF-1 cells under electron microscope asnumerous non-enveloped, homogeneous, spherical to icosahedral particleswith diameter of 20-25 nm (FIG. 7). Some of the viral particles wereincluded in the inclusion bodies and the others could be found in thecytoplasm (FIG. 7). The isolated viral particles could be furtherpurified by CsCl density gradient centrifugation. Using GNNV as anexample, the purified virus was a non-enveloped icosahedral virionparticle with the diameter of 20-25 nm. The buoyant density of GNNV inCsCl was 1.34 g/cm³.

[0078] In addition to the findings of CPE in the GF-1 cells, theexistence of an aquatic virus in the GF-1 cells and the capability ofthe cells to multiply the virus can be further confirmed by fourmethods: (1) the PCR method; (2) the Western immunoblot method; (3) theELISA method; and (4) the immunofluorescent staining method.

[0079] Using GNNV as an example, the PCR method could be accomplished bychoosing a pair of primers, i.e., R3 (SEQ ID NO. 1) and F2 (SEQ ID NO.2), for PCR amplification. The target fragment T4 exists in fishnodavirus. Therefore, the PCR method using F2 and R3 was specific tofish nodavirus, not just GNNV. The results of the PCR study showed thatGNNV could be replicated in the GF-1 cells and released into thesupernatant of culture cells (FIG. 6).

[0080] The Western immunoblot using mouse anti-GNNV serum demonstratedthat viral proteins were present in the GNNV-infected cells cultured at20-32° C., suggesting that the viral mRNA could be successfullytranslated into viral polypeptides within the host cells when theculture was maintained at 20-32° C.

[0081] The ELISA and immunofluorescent staining methods also showedpositive reactions with the anti-GNNV serum, indicating that GNNV couldbe multiplied in the GF-1 cells.

[0082] In accordance with a third embodiment of the present invention,there is provided for an anti-NNV antibody and the method of making theantibody. The experimental designs in this embodiment are illustrated asfollows:

EXAMPLE 3 Production of Anti-NNV Antibodies

[0083] (1) Production of Anti-NNV Antibodies

[0084] Polyclonal antibodies can be produced in accordance withconventional methods, e.g., by sequential injections of the purified NNVimmunogen into a suitable animal such as a rabbit, rat, or mouse. Forexample, a suitable amount of the NNV immunogen can be injectedintravenously, subcutaneously, or intraperitoneally to a rabbit andboosted twice or more at 2 or 3 week intervals. The injection maycontain a suitable amount of Freund's complete or incomplete adjuvant,if necessary.

[0085] For the production of monoclonal antibodies, immunizing mice ispreferred. Three or four days after the final boost, spleen cells ofmice can be separated and fused with myeloma cells, e.g., SP2/0-Ag14myeloma cells (ATCC CRL 1581), in accordance with a conventional methoddescribed by Mishell and Shiigi (Selected Mthods in Cellular Immunology,W. H. Freeman & Company, 1980). The spleen cells and the myeloma cellscan be used in a ratio ranging from 1:1 to 1:4. A fusion-promotingagent, e.g., polyethylene glycol (PEG) 4000, may be employed foraccelerating the cell fusion. A medium suitable for use in the cellfusion step may be RPMI 1640 (Gibco BRL, Life Technologies, Inc.) andthe medium generally contains 10-15% (v/v) fetal bovine serum (FBS).

[0086] The fused cells can be cultured in the RPMI1640-15% FBS,supplemented with hypoxanthine, thymidine and aminopterin, and afterseven to ten days, positive hybridoma clones producing antibodiesspecific for NNV can be selected by ELISA assay using the culturesupernatant. Further selection of positive clones can be accomplished byusing conventional methods, e.g., the limiting dilution technique, theplaque method, spot method, agglutination assay and autoradiographicimmunoassay.

[0087] (2) Purification of Antibodies

[0088] Antibody can be purified by conventional immunoglobulinpurification procedures such as ammonium sulfate precipitation, gelelectrophoresis, dialysis, affinity chromatography, and ultrafiltration.Ion exchange, size exclusion hydroxylapatite, or hydrophobic interactionchromatography can be employed, either alone or in combination. Lightand heavy chain can be carried out using gel electrophoretic techniquesor isoelectric focusing, as well as other techniques known in the art.

[0089] In accordance with a fourth embodiment of the present invention,there is provided a vaccine to NNV and a method for protecting fishagainst NNV infection. The experimental designs in this embodiment areillustrated as follows:

EXAMPLE 4 Production of NNV Vaccines

[0090] Preparation of Vaccine using Killed NNV

[0091] The vaccine of the present invention is administered as a killedvaccine, which encompasses any methods now known or hereafter developedfor killing. The preferable method is by heat treatment. The heattreatment method can be accomplished by heating the purified NNV to atemperature sufficiently to inactivate the virus (such as 70° C.) for asufficient amount of time (such as for 24 hours). After the heating stephas been completed, for intraperitoneal or intramuscular vaccination,the inactivated NNV can be emulsified in Freund's incomplete adjuvant(FIA) using a mixer for several minutes. The vaccine can then beinjected into the fish (the primary injection). Booster injections canbe given to the fish 30-45 days after the primary injection. Normally,the booster injection consists of about one half of the volume of thevaccine used in the primary injection. The fish then can receive asecondary boost 10 days after the first booster shot is administered.The serum samples from the fish at various time points can be taken fortiter determination.

[0092] For orally administered vaccine, an enteric coating containingnon-toxic polymeric materials can be added to the vaccine. Thepreferable enteric coating materials are the ones which can resistdissolution at the pH of the stomach but can be dissolved once thematerial passes from the stomach to the pyloric caecum and intestines.For example, cellulose acetate phthalate, hydroxypropylmethyl cellulosephthalate, carboxymethylethyl cellulose, hydroxypropylmethyl celluloseacetate succinate, cellulose acetate trimellitate, polyvinyl acetatephthalate, EUDAGRIT L-30D and 1100-55, EUDAGRIT L 12.5 and L 100,EUDRAGIT E, RL, RS and NE are among the preferred materials. Additionalmaterials can be used in combination with the enteric coating materials.For instance, plasticizers (such as polyethylene glycol 200, 400, 1000,4000, 6000, propylene glycol, PVPK-90, glycerin or glycerol, diethylphthalate, oleic acid, isopropyl myristate, liquid paraffin or mineraloil, triacetin, glycerol monostearate, dibutyl sebacate, triethylcitrate, tributyl citrate, acetylated monoglyceride, dibutyl phthalate,acetyl tributyl citrate, castor oil, and glycerol tributyrate);disintegrants (such as sodium starch glycolate); adjuvants (such asimmunostimulants [e.g., beta glucan]); binders (such as starch,polyvinyl pyrrolidone, polyvinyl alcohol); diluents (such as lactose);lubricants (such as magnesium stearate) etc. can all be used with theenteric coatings. For oral administration, fish can receive the vaccineon an every-other-day basis for a total of thirty days. The effects ofthe vaccines can be monitored by the use of ELISA.

[0093] Orally administered vaccine is generally the preferred method ofvaccinating fish because it is not limited by the size of the fish thatcan be handled, and it reduces the stress on the fish associated withimmersion and intraperitoneal injection. Furthermore, oral vaccinesoffer the additional advantages of stimulating the gut-associatedlymphoid tissue to a greater extent than does intraperitoneal injection.

[0094] The present invention has been described with reference toseveral preferred embodiments. Other embodiments of the invention willbe apparent to those skilled in the art from the consideration of thisspecification or practice of the invention disclosed herein. It isintended that the specification and examples contained herein beconsidered as exemplary only, with the true scope and spirit of theinvention being indicated by the following claims:

1 2 1 20 DNA Artificial Sequence primer_bind synthetic oligonucleotideprimer complementary to viral sequence 1 cgagtcaaca cgggtgaaga 20 2 20DNA Artificial Sequence primer_bind synthetic oligonucleotide primercomplementary to viral sequence 2 cgtgtcagtc atgtgtcgct 20

What is claimed is:
 1. An immortal cell line derived from grouper whichis susceptible to aquatic viruses.
 2. The immortal cell line of claim 1,wherein said grouper is Epinephelus coioides.
 3. The immortal cell lineof claim 2, which is ATCC deposit No. ______.
 4. The immortal cell lineof claim 3, wherein said cell line is derived from grouper fin tissue.5. The immortal cell line according to claim 4, wherein said cell lineis susceptible to and mass produces a virus.
 6. The immortal cell lineaccording to claim 5, wherein said virus is selected from the groupconsisting of Birnaviridae, Herpesviridae, Reoviridae, and Nodaviridae.7. The immortal cell line according to claim 6, wherein saidBirnaviridae is Infectious Pancreatic Necrosis Virus (IPNV).
 8. Theimmortal cell line according to claim 6, wherein said Herpesviridae isEel Herpes Virus Formosa (EHVF).
 9. The immortal cell line according toclaim 6, wherein said Reoviridae is Hard Clam Reovirus (HCRV).
 10. Theimmortal cell line according to claim 6, wherein said Nodaviridae is afish nodavirus which is selected from the group consisting of NervousNecrosis Virus (NNV), Fish Encephalitis Virus (EFV), Piscine NeuropathyNodavirus (PNN), Grouper Nervous_Necrosis Virus (GNNV), Stripped JackNervous Necrosis Virus (SJNNV), Tiger Puffer Nervous Necrosis Virus(TPNNV), Berfin Flounder Nervous Necrosis Virus (BFNNV) and Red SpottedGrouper Nervous Necrosis Virus (RGNNV).
 11. A method for establishing animmortal cell line comprising: establishing a primary cell culture byplacing cells released from fin tissue of Epinephelus coioides in atissue culture flask until a monolayer of cells is formed; subculturingand maintaining said monolayer of cells in a media suitable for cellsubculturing; and monitoring the transformation of said subculturedcells which is characterized by susceptibility to aquatic viruses.
 12. Amethod for growing a virus comprising the steps of: inoculating saidvirus into an established cell culture comprising the immortal cell lineaccording to claim 1; and incubating said cell culture in a nutrientmedium suitable for growth and replication of said virus.
 13. The methodfor growing a virus according to claim 12, wherein said virus isselected from the group consisting of Birnaviridae, Herpesviridae,Reoviridae, and Nodaviridae.
 14. The method for growing a virusaccording to claim 13, wherein said Birnaviridae is InfectiousPancreatic Necrosis Virus (IPNV).
 15. The method for growing a virusaccording to claim 13, wherein said Herpesviridae is Eel Herpes VirusFormosa (EHVF).
 16. The method for growing a virus according to claim13, wherein said Reoviridae is Hard Clam Reovirus (HCRV).
 17. The methodfor growing a virus according to claim 13, wherein said Nodaviridae is afish nodavirus which is selected from the group consisting of NervousNecrosis Virus (NNV), Fish Encephalitis Virus (EFV), Piscine NeuropathyNodavirus (PNN), Grouper Nervous Necrosis Virus (GNNV), Stripped JackNervous Necrosis Virus (SJNNV), Tiger Puffer Nervous Necrosis Virus(TPNNV), Berfin Flounder Nervous Necrosis Virus (BFNNV) and Red SpottedGrouper Nervous Necrosis Virus (RGNNV).
 18. A method for mass producinga virus comprising: inoculating the virus in the established cellculture comprising the immortal cell line as claimed in claim 1;incubating said cell culture in a nutrient medium suitable for growthand replication of said virus until an appearance of cytopathic effects(CPE); and harvesting said virus from said cell line.
 19. A method forpurifying a virus comprising: oculating the virus into an establishedcell culture comprising the immortal cell line as claimed in claim 1;incubating said cell culture in a nutrient medium suitable for growthand replication of said virus until an appearance of cytopathic effects(CPE); harvesting said virus from said cell line; and purifying saidvirus using a density gradient centrifugation.
 20. The method forpurifying a virus according to claim 19, wherein said density gradientcentrifugation is a CsCl density gradient centrifugation.
 21. The methodfor purifying a virus according to claim 19, wherein said virus is NNV,and wherein said NNV has a buoyant density of approximately 1.34 g/ml inCsCl.
 22. A method for detecting a virus in an immortal cell linecomprising: observing a development of cytopathic effects (CPE) in anestablished cell culture comprising the immortal cell line according toclaim 1 under a microscope.
 23. The method for detecting a virus in animmortal cell line according to claim 22, further comprising the stepsof: fixing said cell line in glutaraldehyde and osmium tetraoxide;detecting viral particles in an ultrathin section of said fixed cellline under an electron microscope.
 24. A method for detecting NNV in animmortal cell line comprising: observing a development of cytopathiceffects (CPE) in an established cell culture comprising the cell lineaccording to claim 1 under a microscope; extracting a viral RNA from thecell culture; and amplifying said viral RNA by PCR method using areverse primer having the DNA sequence of SEQ ID NO. 1 and a forwardprimer having the DNA sequence of SEQ ID NO. 2 .
 25. A method fordetecting NNV in an immortal cell line comprising: observing adevelopment of cytopathic effects (CPE) in en established cell culturecomprising the cell line according to claim 1 under a microscope;extracting a viral protein from the cell line; electrophoresizing saidviral protein in an SDS-polyacrylamide gel; and analyzing saidpolyacrylamide gel by western immunoblot using an anti-NNV serum.
 26. Amethod for detecting NNV in an immortal cell line comprising: observinga development of cytopathic effects (CPE) in an established cell culturecomprising the cell line according to claim 1 under a microscope;extracting NNV protein from said cell line; and detecting NNV usingELISA.
 27. A method for detecting NNV in an immortal cell linecomprising: fixing said cell line according to claim 1 in formalin;adding a mouse anti-NNV antibody serum to the cell line; and stainingsaid cell line with flouroscein isothiocynate (FITC) conjugated goatanti-mouse antibody.
 28. An anti-NNV antibody comprising: an anti-NNVserum which is produced by administering an effective amount of NNVwhich is purified according to claim 21 to a suitable animal tostimulate the immunoresponse of said animal.
 29. The anti-NNV antibodyaccording to claim 28, wherein said antibody is a monoclonal antibody.30. A method for preparing an anti-NNV antibody comprising: injecting aneffective amount of NNV which is purified according to claim 21 to asuitable animal to stimulate an immunoresponse; and collecting andpurifying a serum from said animal.
 31. A vaccine for protecting fishagainst NNV infection comprising: an immunogenically effective amount ofkilled NNV which is purified according to claim
 21. 32. The vaccineaccording to claim 31, further comprising one or more materials selectedfrom the group consisting of adjuvants, plasticizers, pharmaceuticalexcipients, carriers, diluents, binders, lubricants, glidants, andaesthetic compounds.
 33. The vaccine according to claim 31, wherein saidvaccine is orally administered.
 34. The vaccine according to claim 33,wherein said orally administered vaccine further comprises an entericcoating, and wherein said enteric coating is impervious to dissolutionin the stomachs of fish.
 35. The vaccine according to claim 31, whereinsaid vaccine is provided by injection.
 36. A method for protecting fishagainst NNV infection comprising: providing a vaccine produced accordingto claim 31 to a fish.